JPS60120823A - Igg monomer - Google Patents

Abstract

PURPOSE:An IgG, obtained by treating an IgG derived from humans with an acid under specific conditions, and removing IgG and/or polymer, and capable of intravenous injection. CONSTITUTION:An IgG monomer obtained by oxidizing 0.2-20wt%/vol% solution of human IgG containing human IgG dimer and/or polymer (IgG agglutination type) as an impurity at 3.7-4.3pH and 1-10 deg.C for 30min-20hr with an acid, e.g. an inorganic acid such as hydrochloric acid or an organic acid such as acetic acid. In the treatement with the acid, an inorganic salt, saccharide, protein and an organic acid salt, etc. alone or plural may be preferably added to prevent the denaturation of the IgG. The resultant IgG treated with the acid is subjected to germ-free filtration and freeze drying, etc. to afford the aimed IgG pharmaceutical capable of intravenous injection. The pharmaceutical contains undenatured IgG monomer, and is very useful due to no formation of IgG agglutination type even on returning to the neutral state.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to intravenously injectable IgG. More specifically, a vein obtained by treating human-derived 1gG with acid under specific conditions to remove IgG dimers and/or polymers (hereinafter, these may be collectively referred to as IgG aggregates) Concerning IgG that can be used.

IgG produced by cold ethanol fractionation, ammonium sulfate fractionation, etc.
In addition to 7SIgG (7), the preparation contains 10-40% ■g
Contains G-aggregated type. This substance cannot be administered intravenously because it exhibits anti-complement activating effects and sometimes antigenicity. In order to produce ■gG for intravenous injection, these I
It is necessary to remove the gG aggregate type.

Regarding the method of removing aggregated TgG, there are the following prior art techniques regarding acid treatment.

It has been observed by Kochwa et al. (1966) that the rigid form of myeloma RGG dissociates upon pH 4 treatment. Reported to repolymerize.

11ansson (196B) is a normal human IgG
It has been reported that when the IgG is adjusted to pl+3.0 without dialysis, the aggregated IgG contained therein dissociates, but when it is returned to neutrality, insoluble matter is formed. In this way, I
Although it has been known that aggregated gG dissociates, there have been no detailed reports on a method for efficiently obtaining monomeric 1gG.

On the other hand, regarding acid denaturation of human IgG, Jirgenso
Although there are reports of observations of optical rotation, circular dichroism, etc. by N. et al. (954) and Doi et al. (1970), there are no reports on how other biological properties change. On the other hand, 5tol111r et al. (1976) and 4nkelhake et al. (1980) conducted research on acid denaturation using rabbit 1[G. lS on the side
, and that the antigen binding ability was stable even at pH 2.5 to 3.0. However, rabbit Ig
Since G has different properties from human IgG, it is difficult to say that such knowledge applies to human IgG.

If we could find a way to dissociate the aggregated form of human IgC without denaturing the human monomer, it would be possible to efficiently produce monomeric rich intravenous globulin from conventional intramuscular globulin ( It becomes possible to manufacture.

Therefore, the first object of the present invention is to provide human IgG that can be injected intravenously.

A second object of the present invention is to provide a method for producing human IgG containing as little polymerized 1gG as possible.

The third object of the present invention is to provide human IgG that does not cause IgG&aggregation even in neutral conditions.

A fourth object of the present invention is to provide a stable, intravenously injectable human IgG.

The present invention provides a method for preparing human 1gG containing human IgG polymers.
．． 2-20 w/v% warm solution pH 3,7-I1.3
It consists of an IgG that can be administered intravenously and is obtained by dissociating the IgG conjugate into an intermediate form by treating it for 30 minutes to 20 hours.

The human IgG used as the raw material is selected from rgG condensed nest type. If it is highly purified, it can be prepared as a drug immediately after the treatment of the present invention, and if it is in the crude stage, it can be combined with the known Ifi production method. As a method for preparing raw human IgG, an alcohol fractionation method using cold ethanol is suitably used (Journal of Clinical Investigation, L. Tun, 417.1944).
Journal of the American Chemical Society, AE, 479.1946).

The acid treatment is performed while keeping the raw material at pH 3.7 to 4.3. The treatment temperature is 1 to 10°C, preferably 3 to 6°C, and the treatment time is 30 minutes to 20 hours, preferably 60 minutes to 4 hours, and more preferably 120 minutes to 200 minutes. pog here
The acid used for the I&I adjustment may be any acid that can be adjusted to ρ11 and does not adversely affect RGC, such as inorganic acids such as hydrochloric acid and organic acids such as acetic acid. The concentration of IgG during acid treatment was 0.
．． It is 2 to 20 w/v%, preferably 10 to 18 w/v%.

In order to prevent denaturation of IgG during this acid treatment, it is preferable to add one or more compounds selected from inorganic salts, saccharides, proteins, and ionic acid salts. Inorganic salts include salts of halogens and alkali metals such as sodium chloride and potassium chloride; sugars include glucose, fructose, nlubitol, mannitol, and saccharose; proteins include albumin and gelatin; organic acid salts include oxalates, citrates (especially alkali metal salts such as sodium salts and potassium salts of these organic acids)
etc. are suitably exemplified. The total amount added is 5 to 2
It is 0W/V%.

From the viewpoint of storage stability of IgG, it is preferable that the stabilizer is allowed to exist as it is even after the production of 1gG of the present invention.

The acid-treated 1gG thus obtained can also be made into a freeze-dried preparation by performing sterilization filtration and freeze-drying according to conventional methods for pharmaceutical mold manufacturing.

Intravenous administration adjusted under specific conditions according to the present invention 1
[G is because the IgG monomer is not denatured, and even if it is returned to neutrality, IgG aggregation will not occur.
It is extremely useful.

Experimental examples and examples of the present invention are shown below.

Experimental Example 1 5 and 7 mg/ml IgG aggregation aggregation aqueous solutions were adjusted to various p+1, incubated at 28° C. for 1 minute, and then neutralized by adding Na01+. Furthermore, an equal volume of phosphate buffer was added, and the amount of residual 1gG aggregate and the degree of plasmin digestion were measured by high performance liquid chromatography. The sample solution used here contained 38% 7S and 61% dimer.
and 1% polymer. When this material was treated with an acid, m became a peak of 78 in the treatment at pH 3 and 8. Dimer 1gG also decreased with decreasing treatment pH, but the amount of polymer increased in treatment under ρ 113.81ff.

The extent of plasmin digestion was slowest for treatments near pH 4, and digestion by plasmin was faster in treated areas where polymer increased and where dimer remained.

From the above, it is clear that the product obtained by the treatment of the present invention has a high monomer content and that IgG aggregation does not occur through neutralization.

Experimental Example 2 Aqueous solution containing 1.4+ng/ml of IgG aggregate type (7S
(38% dimer, 61% dimer, 1% polymer), various stabilizers were added, pl+ was adjusted to 3.8, and after 1 incubation at 28° C. for 60 minutes, Na0II was added to neutralize. Then, the measles antibody titer of IgG was measured using Hemaggluti++.
It was measured by the ++lion 1nhibition test method and expressed in international units (IU/100mg) to examine the effect of the stabilizer. The results are as follows.

:　W　v%　-”L kara 1 bat price- Su 7 Albumin: ] 5 10 Same as above: 1 lO Gelatin: 10 1O NaCI: 15 10 Same as above = 19 Glucose, 15 10 Same as above: 19 Saccharose 8t0 10 Mannitol: 15t.

1 1 10 Na citrate: 15 10 1 9 NaC 1:5 10 Fructose = 5 10 Example 1 15 W/V% of 1g G of HiL-1 obtained by alcohol fractionation
The solution was adjusted to pH 4.0 using 0.1N hydrochloric acid,
It was left standing at 4°C for 3 hours. Thereafter, it was corrected to 1116.5 using 0.1N caustic soda. Then NaCl
IOW/V% and glucose were added to the mixture to give a concentration of 5w/v%, and the mixture was sterilized and filtered to obtain globulin for intravenous injection.

Patent Applicant: Midori Juji Procedure Co., Ltd. NeTit Positive Side: (Voluntary) 1. Indication of the case: Patent Application No. 228638, filed in 1982. 2. Name of the invention: TgG monomer. 3. Person making the amendment. Relationship with the case: Patent Applicant name (name) Midori Juji Co., Ltd. 4, agent ■541 Address 4-1'-153 FLR, Hirano-cho, Higashi-ku, Osaka-shi 3
New Life Hirano-cho 40 (No. 5 Telephone (06) 227-1156 6. "Claims" column and "Detailed description of the invention" column 7 of the specification to be amended, Contents of the amendment (1) Specification ``Claim 1 is amended as shown in the attached sheet.''

(2) Add "citric acid, oxalic acid" next to "acetic acid" on page 5, line 19 of the specification.

(3) In the same book, page 6, line 6, add ``and sodium phosphate'' next to ``salt with a metal.''

(4) Delete "Add rNaOll" on page 8, line 10 of the same book.

(5) "Albumin: 15" on page 8, line 17 of the same book is corrected to "albumin: 5".

(6) "Mannitol: 15" on page 9, line 5 of the same book is corrected to "mannitol: 5."

(7) Add [albumin; 1 1-glycine: 2.5 10J] between line 10 and line 1-1 on page 9 of the same book.

(8) “0,1N hydrochloric acid” on page 9, line 13 of the same book is r
O, IM vinegar #i loose 1 pot i liquid (all 3.5) Correct to J.

(9) Add "0.1N caustic soda" on page 9, lines 14-15 to r1M glycine buffer (all 9.5
) Correct to J.

(10) On page 9 of the same book, line 15, after “after that,” [
Add albumin at 1 w/v%.

(Eng. 1) "rjOw/v%" on page 9, line 16 of the same book is r
Corrected to 3 w/v%.

Scope of claims +1. l Hi! -1gG dimer and/or IF)
0.2-2Q of TgG w/
% solution at pH 3.7 to 4.3 for 30 minutes to 20 hours to decompose H] and [gG dimers and polymers into small molecules.
Intravenously administrable IgG obtained by illumination.

(2) Claim No. 11 characterized in that the stabilizer selected from inorganic salts, sugars, proteins, and organic acid salts is dissociated in the presence of a concentration of 0.5 to 20 w/v%. (3) The inorganic salt is sodium chloride, sodium phosphate, or potassium chloride, and the ti7 kB is glucose,
The intravenously administrable product according to claim (2), which is fructose, zasocalose, sorbitol or mannitol, the protein is albumin or gelatin, and the organic acid salt is oxalate or citrate. NargG.

Claims (1)

[Claims] fil A 0.2 to 20 w/v% solution of human IgG contaminated with human IgG dimers and/or polymers was prepared at pH 3.
, 7-4.3 for 30 minutes to 20 hours to dissociate human 1gG dimers and polymers into monomers, which can be administered intravenously. (2) Dissociated in the presence of at least one stabilizer selected from inorganic salts, saccharides, proteins, and organic acid salts at a concentration of 0.5 to 20 W/■% Claim fi1 Intravenously administrable IgG0 (3) The inorganic salt is sodium chloride or potassium chloride, and the saccharide is glucose, fructose, sucrose,
Sorbitol or mannitol, the protein is albumin or gelatin, and the organic acid salt is oxalate,
Claim i, characterized in that it is a citrate salt.
The method according to item 11.